Hypoglycemic and anti-diabetic activity of ethanolic extract of Catharanthus pusillus (murray) g.don

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1 IOSR Journal of Pharmacy ISSN: , Volume 2 Issue 4 July-August 212 PP Hypoglycemic and anti-diabetic activity of of Catharanthus pusillus (murray) g.don A. Navitha 1, D. A. Helen Sheeba 2, C. Ramesh 3, M. Sartaj Banu 4 1, 2, 4 (Department of Pharmacology, Malla Reddy Institute of Pharmaceutical Sciences, Hyderabad 57, India) 3(Department of Pharmacology, GSN Pharmaceuticals Pvt. Ltd., Kukatpally, Hyderabad- 572, India) Abstract Many plant drugs are studied for effecting in the treatment of Diabetes mellitus. In the present study of herb of Catharanthus pusillus was evaluated for hypoglycemic effect in Streptozotocin induced diabetic rats using both 18 hour fasted rat model and oral glucose tolerance test. A comparison was made between the actions of Catharanthus Pusillus and known anti diabetic drug Glibenclamide (5 mg/kg, p.o). The of Catharanthus Pusillus was administered at different doses to normal and diabetic rats. The at 5 mg/kg body weight dose level exhibited significant hypoglycemic activity (P <.1 and P <.1). Phytochemical screening of aqueous, methanolic, ethanolic and chloroform extract revealed the presence of alkaloids, saponins and carbohydrates. It is concluded that of herb of Catharanthus Pusillus posses anti diabetic activity. Keywords Hypoglycemic activity, Oral glucose tolerance test, Catharanthus Pusillus Streptozotocin induced Diabetes. I. INTRODUCTION Non insulin dependent diabetes mellitus, type 2 (NIDDM) can be manifested by chronic hyperglycemia due to defects in insulin secretion, insulin action and or both [16]. It is common disorder among the Indian population. Therapeutic options for diabetes are diet, exercise, oral hypoglycemic drugs and insulin therapy. In India number of alternative medicines like Ayurvedic as well as Sidha preparations have attracted great interest in type 2 diabetes mellitus management [2], Catharanthus Pusillus (C. Pusillus) is one such Ayurvedic herbal formulation. Currently 17 million people world wise affected by diabetes mellitus. Statistical projections about India suggested that 57 million Indians will be affected by diabetes mellitus by the year 225 making the country with highest number of diabetics in the world [1]. Catharanthus Pusillus belonging to family Apocynaceae is known with various names in India and all over the world. In India seven flowers/ leaves of Catharanthus species are used at a time whereas in the Cook Islands 18 leaves boiled in a kettle of water and in the west indies roots of plants infused in whiskey are used traditionally [3]. In the present study we have evaluated Hypoglycemic activity in of herb of Catharanthus Pusillus and the comparison was made between the actions of Catharanthus Pusillus and anti-diabetic drug Glibenclamide. II. MATERIALS AND METHODS 2.1 Plant Material Herb of Catharanthus Pusillus [12] was collected after authentication by Dr. K. Madhava chetty, assistant professor, department of botany, Sri Venkateswara University, Tirupati, Andhra Pradesh, India. The plant was collected from the chitturu forest; collected plant was washed thoroughly with water and dreid in the shade. Dreid plant was made into powder in a grinder and were extracted with 1 volumes of ethanol and macerated at room temperature in ethanol for 24 hours. The extract was suctioned and filtered using whatmann filter paper. This was repeated for two more days and similar extracts were pooled together and concentrated at 4 degrees under reduced pressure using buchi R-153 rotavapour. The was used for further study. 2.2 Experimental Animals The experiments were performed in adult albino wister rats weighing from 2-24gm. The animals maintained at 23+1 degrees, 55+5% humidity, 12 hours light and 12 hours dark cycle. Animals had free access to diet and water followed by standard laboratory animal diet conditions [6]. 17

2 Hypoglycemic and Anti-diabetic activity of Ethanolic extract of Catharanthus 2.3 Chemicals Streptozotocin purchased from SISCO research laboratories Pvt. Ltd, Mumbai. All chemicals used for this study were analytical grade. 2.4 Study on Glucose Loaded Animals (Oral Glucose Tolerance Test {Ogtt}) The OGTT was performed on overnight fasted animals (18 hours) normal rats. Animals divided into three groups, those are Group I 5 % tween 8 treated (Control). Group II Catharanthus Pusillus treated (25mg/kg body weight). Group III Catharanthus Pusillus treated (5mg/kg body weight)..5 ml of 5% tween 8 administered to the control group whereas group II treated with 25 mg/kg body weight of of Catharanthus Pusillus and group III treated with 5mg/kg body weight of ethanolic extract of Catharanthus Pusillus respectively. Glucose ( 1 gm/kg ) was fed 3 min. after pretreatment with.5 ml of 5% tween 8 and of Catharanthus Pusillus Blood glucose levels were measured immediately drawing the blood from tail vein at, 6, 12, 18, 3 minutes after glucose load to access the effect of extract on blood glucose levels of glucose loaded animals. Blood glucose levels were determined by the glucose oxidase method using glucometer ( Accuchek active) [9]. Results were tabulated in table. 1 The percent induced glycemia (%IG) following oral glucose load at different time intervals was calculated for the control and treated groups as follows: Gx Go % IG = Go Where Go is the initial glycemia (mg/dl) and Gx the glycemia (mg/dl) at different time intervals after the oral glucose load. 2.5 Induction of Diabetes Initial determination of 12 hours fasting blood glucose levels, blood drawn from tail vein. After determination animals were given single I.P injection of streptozotocin at dose 5 mg/kg (freshly dissolved in physiological saline) and blood levels of each rat was determined after 72 hours Streptozotocin (STZ) administration for the confirmation of diabetes. Rats with blood glucose level above 25 mg/dl were considered as diabetics and used subsequently [6]. 2.6 Hypoglycemic Activity Hypoglycemic activity performed on overnight fasted (18 hours) 3 rats (24 diabetic rats and 6 normal rats). The animals divided into five groups, those are Group I Normal. Group II Diabetic control Group III - Glibenclamide (5 mg/kg, p.o). Group IV - 25 mg/kg body weight of of Catharanthus Pusillus. Group V - 5 mg/kg body weight of of Catharanthus Pusillus. Group I were administered orally with 2% gum acacia (vehicle) whereas group II received streptozotocin, group III received glibenclamide (5 mg/kg, p.o) and group III and IV were administered with 25 mg/kg and 5 mg/kg body weight of of Catharanthus Pusillus suspended in 2% gum acacia for 14 consecutive days. The animal dose has been derived from the human dose by using standard dose ratios. The blood samples collected from the tail vein of rats immediately before and 1, 5, 7 and 14 days after administration of formulation. The blood glucose levels were determined by the glucose oxidase method using glucometer (Accucheck active) [9]. Results were tabulated in table Statistical Analysis All the values were expressed as mean ± SEM (standard error mean) n= 6 rats. The differences were compared using one way analysis of variance (ANOVA) followed by Dunnett s t test. P values <.1,.1 and.5 were considered as significant. III. RESULTS 3.1 Effect of of catharanthus pusillus on glucose loaded rats The were subjected to screening for anti hyperglycemic activity by oral glucose tolerance test in albino rats and both 25 mg/kg and 5 mg/kg body weight doses possessed significant antihyperglycemic activity the blood glucose levels are showed in table 1. 18

3 Blood glucose lvels Blood glucose levels S.No Drug/Control Body Weight 1 5 % tween 8 (Control) Hypoglycemic and Anti-diabetic activity of Ethanolic extract of Catharanthus TABLE. 1 Blood Glucose Level mg/dl hr 1 hr 2 hr 3 hr 5 hr 24 hr 142±2.8 8± ± ± ±2.6 16±5.4 98± mg/kg Body Weight of of C.Pusillus 3 5 mg/kg Body Weight of of C.Pusillus 17±3.9 96± ±4.3 ** 122±1.7 ** 115±2.4 ** 111±3.2 * ±5.9 * 15±5.4 98± ±1.2 ** 18±2.9 ** 85±1.8 ** 7±4.6 * 55±2.4 * Values are represented as mean ± SEM (n=6 rats). Values are statistically significant at *P <.5. Ethanolic extract of C. Pusillus compared with 5% tween 8 treated rats. The of C. Pusillus at 5 mg/kg body weight dose level exhibited significant hypoglycemic activity shown in Fig.1. Figure Hours 5% tween 8 25 mg/kg E.E of C. Pusillus 5 mg/kg E.E of C. Pusillus Fig.1. Effect of Catharanthus Pusillus on Glucose loaded Rats OGTTmodel The percentage reduction of blood glucose levels at 2 hrs is 43.4% shown by 25 mg/kg and 78.2% shown by 5 mg/kg body weight of of C.Pusillus. These compared with 5% tween 8 glucose loaded rats. The reduction of blood glucose levels are shown in Fig Hours 5% tween 8 25 mg/kg E.E of C. Pusillus 5 mg/kg E.E of C. Pusillus Fig.2. Effect of Catharanthus Pusillus on Blood Glucose levels of albino Rats 19

4 Blood glucose levels mg/dl Hypoglycemic and Anti-diabetic activity of Ethanolic extract of Catharanthus 3.2 Effect of of catharanthus pusillus on diabetic rats The results of the present study demonstrate that the of Catharanthus puillus had a significant hypoglycemic effect and anti-diabetic activity in streptozotocin induced rat. The blood glucose levels are showed in table. 2. TABLE. 2 Groups days 1 days 5 days 7 days 14 days Normal. 89.2±2.5 87±1.4 88±2.2 86± ±3.6 Diabetic control 274±5.5 27± ±4.2 28± ±2.6 Streptozotocin + Glibenclamide 25 mg/kg body weight of of Catharanthus Pusillus. 5 mg/kg body weight of of C. Pusillus 28±4.4 26±5.6 * 245±3.2 * 25±2.8 ** 181.2±1.9 * 326.3±6.4 28±2.6 * 261.2±4.2 ** 246±2.9 ** 21±3.8 *** 394±4.2 32±3.6 *** 285±3.2 *** 254±1.7 * 25±4.9 ** Values are represented as Mean ± SEM (n=6 rats). Values are statistically significant at ** P <.1, *** P <.1. Diabetic + Catharanthus Pusillus (25 mg/kg, 5 mg/kg) compared with diabetic + glibenclamide and Normal control rats. The of C. Pusillus showed significant anti-diabetic activity in Fig days 1 days 5 days 7 days 14 days Normal. Diabetic control Glibenclamide 25 mg/kg B.W of E. E. of C.Pusillus. STZ + 5 mg/kg B.W of E. E. of C. Pusillus Fig.3. Effect of Catharanthus Pusillus on Fasting blood glucose levels of albino rats IV. DISCUSSION The of Catharanthus Pusillus at 25 mg/kg and 5mg/kg showed significant antidiabetic activity when compared normal, control and diabetic rats. The anti-diabetic property of ethanolic extract of herb of Catharanthus Pusillus exhibited has dose dependent activity. The anti-diabetic effect of of herb of Catharanthus Pusillus at the dose of 5 mg/kg is compared to the standard drug glibenclamide (5 mg/kg). Our results are supporting it as medicine for the treatment of diabetes. Streptozotocin is probably the most widely used agents producing insulin dependent diabetes mellitus and non insulin dependent diabetes mellitus in experimental animals. Streptozotocin is a glucosamine nitrosourea compound [15]. Streptozotocin enters the B cell via a glucose transporter (GLUT2) and causes 2

5 Hypoglycemic and Anti-diabetic activity of Ethanolic extract of Catharanthus alkylation of DNA. DNA damage induces activation of poly ADP-ribosylation, a process that is more important for the diabetogenicity of streptozotocin than DNA damage itself. Poly ADP-ribosylation leads to depletion of cellular NAD+ and ATP. Enhanced ATP dephosphorylation after streptozotocin treatment supplies a substrate for xanthine oxidase resulting in the formation of superoxide radicals. Consequently, hydrogen peroxide and hydroxyl radicals are also generated. Furthermore, streptozotocin liberates toxic amounts of nitric oxide that inhibits aconitase activity and participates in DNA damage. As a result of the streptozotocin action, B cells undergo the destruction by necrosis [13]. These also exhibit diabetic complications such as myocardial [8], cardiovascular [5], gastrointestinal [1], tracheal [4], kidney [11], urinary bladder [14] and connective tissue [7] dysfunctions similar to diabetic patients. Streptozotocin induced non insulin dependent diabetes mellitus in rats constitute an invaluable pharmacological tool for screening for anti-diabetic activity because most of the diabetic patients are non insulin dependent in nature. V. CONCLUSIONS The Catharanthus pusillus possesses both hypoglycaemic and anti-diabetic activities in normoglycaemic and STZ diabetic rats, respectively. The whole plant extracts did not show a consistent effect on normal blood sugar levels but it effectively reversed the Streptozotocin induced changes in the blood sugar level and the beta-cell population in the pancreas. It also showed a protective effect when it was given prior to Streptozotocin administration. From the above discussion it conclude that whole plant of Catharanthus puillus at high dose (5 mg/kg) exhibited significant anti-hyperglycemic activity than whole plant extract at low dose (3 mg/kg) in Streptozotocin-induced diabetic rats. 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